The hepatic asialoglycoprotein receptor (ASGP-R) was the first endocytic, recycling receptor to be identified and characterized. There are now at least 9 receptors of this type, whose function is to remove a variety of circulating ligands. although these receptors have been studied extensively, many details about how they function are still unknown. For example, the mechanism by which endocytosed ligand and receptor are segregated from each other has not been elucidated. Our long-term goal is to understand the molecular basis for segregation and other steps along the receptor recycling pathway for the ASGP and other receptors. We have discovered that the internalized ASGP-R undergoes an inactivation/reactivation (I/R) cycle as it traverses its intracellular recycling itinerary. Our central hypothesis is that transient ASGP-R inactivation is what makes the segregation step (and thereby the whole endocytic process) so efficient. Since dissociated ligand cannot rebind to inactive ASGP-R, it will be delivered to lysosomes rather than being recycled to the cell surface. We have now succeeded in reconstituting both ASGP-R inactivation and reactivation (the I/R cycle) in an in vitro permeable cell system. ASGP-R inactivation requires only exogenous ATP, while ASGP-R reactivation requires acyl-CoA. The latter exciting finding is the basis for much of this project. Our Specific Aims are: 1. To characterize the inactivation and reactivation of ASGP-Rs in permeable hepatocytes. The kinetics, requirement for cofactors and specificity for fatty acid chain length will be examined. 2. To determine if, and at what site(s), subunits of the ASGP-R are fatty acid acylated. 3H- or 14C-Palmitate will be used to assess covalent modification of the ASGP-R during the I/R cycle. Acylated sites will be defined using HPLC, GLC, mass spectrometry and amino acid sequence analyses of proteolytic fragments. 3. To determine if receptor I/R cycles occur in other endocytic, recycling receptor systems. Eight other similar receptor systems will be assessed for the occurrence of an I/R cycle using various cell types and the appropriate ligands. 4. To determine whether the loss of ASGP-R activity associated with diabetes or chronic alcohol consumption in rats is due to a perturbation of the normal I/R cycle. If we confirm our hypothesis that the ASGP-R I/R cycle is altered in these diseases, then other diseases may be associated with other malfunctioning recycling receptor systems as well. These studies may uncover a new class of pathologies related to the inability of different endocytic receptors to remove and degrade their respective ligands.